The present invention relates to the field of biology and of the regulation of the signal transduction pathways which respond to extracellular stimuli. More specifically, the present invention is concerned with novel polypeptides which are derived from the human JNK3 protein, their variants, the corresponding nucleotide sequences, and their uses.
The protein JNK (c-jun N-terminal kinase), also termed SAPK (stress-activated protein kinase), belongs to the family of MAP (mitogen-activated protein) kinases. It is involved in the signal transduction pathways which respond to extracellular stimuli (for example, proinflammatory cytokines or environmental stresses). Its activation requires the phosphorylation of threonine 221 and tyrosine 223 within a highly conserved T-P-Y tripeptide motif located in the kinase domain (Dxc3xa9rijard et al. 1994). The substrates of JNK are transcription factors such as c-jun (phosphorylated on serine 63 and serine 73), ATF-2 (phosphorylated on threonine 69 and threonine 71) and Elk-1.
The JNK proteins exhibit a large number of isoforms, and ten isoforms have been recorded to date. They derive from three different genes, termed JNK1, JNK2 and JNK3. The JNK1 and JNK2 genes encode two isoforms, xcex1 and xcex2, by means of alternative splicing (Gupta et al. 1996). For each isoform, xcex1 and xcex2, there exists a short version and a version which is elongated C-terminally. The short version is linked to the insertion, during the alternative splicing, of five nucleotides which supply a termination codon.
The proteins JNK3xcex11 and JNK3xcex12 are the only two isoforms of JNK3 which have so far been recorded. They differ from JNK1 or JNK2 by, inter alia, an N-terminal extension of 38 amino acids whose function has not yet been established. The rat JNK3 homologue, termed SAPKxcex2, does not exhibit this distinctive feature.
The tissue distribution of the isoforms is very diverse, with variable levels of expression. However, it has been demonstrated that, while the JNK3 isoforms are more selectively expressed in the brain (for example in the hippocampus or in the cerebellum (Mohit et al. 1995)), they are also expressed in the heart and the testes.
While a growing number of results underline the importance of JNK3 in the phenomena of neurodegeneration and neuronal death by apoptosis, the mode of action of JNK3 remains unknown.
It has been shown that the neurones of the CA1 region of the hippocampus of patients who have suffered a period of hypoxia exhibit strong JNK3 immunoreactivity within the nuclei whereas JNK3 immunoreactivity is diffuse and exclusively cytoplasmic in control samples (Zhang et al. 1998). Furthermore, deletion of the JNK3 gene in mice results in resistance to kainic acid, which is an agonist of the glutamate receptors involved in the phenomena of excitotoxicity. The authors (Yang et al. 1997) provide a detailed description of the reduction in the epileptic effects and the prevention of the neuronal death by apoptosis in the hippocampus following injection of kainic acid into these mice which have been deleted for JNK3. Lastly, one of the preferred substrates of JNK3 is the transcription factor c-Jun, which is one of the components of the AP1 complex, which is itself heavily involved in functions of survival and neuronal degeneration. The c-Jun factor appears to have a double function i.e. both in cell death and in neuronal protection (Herdegen et al. 1997). Suppression of the expression of c-Jun or inhibition of its function protects the hippocampal and sympathetic neurones from cell death in culture (Estus et al. 1994, Ham et al. 1995). Finally, while expression of c-Jun is increased in apoptotic neurones which are degenerating following ischaemia, nerve section or irradiation, it is also increased in biopsies taken from patients afflicted with neurodegenerative pathologies such as multiple sclerosis, amyotrophic lateral sclerosis and Alzheimer""s disease, Parkinson""s disease and Huntington""s disease (Anderson et al. 1994, Herdegen et al. 1998, Martin et al. 1996).
While the JNK3 protein kinase nowadays appears to be one of the key elements involved in neuronal degeneration, the precise nature of its mode of action is unknown. In this regard, the identification of new natural isoforms of JNK3 represents a major challenge for understanding the mechanism of action of this protein in the phenomena of neuronal degeneration and for identifying novel targets for aiming at therapeutically.
The present invention describes the detection, cloning and characterization of novel polypeptides derived from JNK3.
The present invention results from characterizing three novel isoforms of human JNK3 termed hJNK3xcex1139, JNK3xcex94Nxcex11 and JNK3xcex94Nxcex12. It ensues, more particularly, from the demonstration that two of these isoforms, i.e. JNK3xcex94Nxcex11 and JNK3xcex94Nxcex12, lack the N-terminal extension which characterizes the known isoforms of JNK3 and the demonstration that these isoforms JNK3xcex94Nxcex11 and JNK3xcex94Nxcex12 exhibit properties which are different from those of the previously described isoforms of JNK3. It furthermore ensues from the discovery that these novel isoforms which lack the N-terminal extension unexpectedly share properties in common with the JNK1xcex21 and JNK2xcex11 isoforms.
The identification of these novel isoforms of JNK3 makes it possible to envisage a large number of applications. These applications cover, in particular, identifying novel neuroprotective compounds which are able to interact specifically with these isoforms. These compounds can be used for preventing and treating different pathologies which are brought about by neuronal degeneration, among which may be mentioned Alzheimer""s disease, Huntington""s disease and Parkinson""s disease, senile dementia and dementia due to AIDS, cranial traumas, cerebral oedemas, hypoxias and anoxias. These novel isoforms can also be used in molecular modelling for achieving an improved understanding of the structure and function of these enzymes and their involvement in pathologies which implicate one or more isoforms of JNK3. Finally, these isoforms of JNK3 are also useful for detecting novel proteins which are involved in intracellular signalling pathways which are specific to each of them. It is thus possible to identify novel relevant targets which are involved in degenerative neuropathologies.
The invention firstly relates to polypeptides which are derived from the JNK3xcex11 or JNK3xcex12 isoforms and which contain an N-terminal or C-terminal deletion. According to a first embodiment, the derivatives are derivatives which contain an N-terminal deletion corresponding to the first 38 amino acids of these isoforms. According to another variant, the derivatives are derivatives which contain a deletion of the C-terminal amino acids from amino acid 139 onwards.
Preferably, the derivatives according to the invention are polypeptides which are selected from the sequences SEQ ID No. 23, SEQ ID No. 25 and SEQ ID No. 27, or a variant of these sequences.
Within the meaning of the invention, the term variant refers to any polypeptide whose structure differs from a polypeptide selected from the sequences SEQ ID No. 23 or SEQ ID No. 25 or SEQ ID No. 27 by one or more modifications of a genetic, biochemical and/or chemical nature and which preserves at least one of the biological properties of said polypeptide. The modification can, in particular, be any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives can be generated for different purposes, such as, in particular, that of improving their level of production, that of increasing their resistance to proteases or of improving their passage across cell membranes, that of increasing their therapeutic efficacy or of reducing their side effects, that of increasing the affinity of the polypeptides for their sites of interaction, or that of conferring novel pharmacokinetic and/or biological properties on it. Advantageously, the variants comprise deletions or mutations affecting amino acids whose presence does not play a decisive role in the activity of the derivative. Such amino acids can be identified, for example, by means of cell activity tests as described in the examples.
The invention also relates to a polypeptide as defined in accordance with the sequence SEQ ID No. 29 and which corresponds to the 1-38 fragment of the of the JNK3xcex11 or JNK3xcex12 forms.
The present invention furthermore relates to any nucleic acid which encodes a polypeptide derived from JNK3, as defined above.
The nucleic acid according to the invention can be a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). Furthermore, it can be a genomic DNA (gDNA) or a complementary DNA (cDNA). The nucleic acid can be of human, animal, viral, synthetic or semisynthetic origin. It can be obtained in different ways, in particular by chemical synthesis using the sequences presented in the application and, for example, a nucleic acid synthesizer. The nucleic acid can also be obtained by screening libraries with specific probes, in particular those described in the application. It can also be obtained by means of mixed techniques including the chemical modification (elongation, deletion, substitution, etc.) of screened sequences obtained from libraries. Generally speaking, the nucleic acids of the invention can be prepared using any technique known to the skilled person.
Preferably, the nucleic acid according to the invention is a cDNA or an RNA. The nucleic acid according to the invention is advantageously selected from:
(a) all or part of the sequence SEQ ID No. 22 or SEQ ID No. 24 or SEQ ID No. 26, or their complementary strand,
(b) any sequence which hybridizes with the (a) sequences and which encodes a derivative according to the invention,
(c) the variants of (a) and (b) which result from the degeneracy of the genetic code.
The nucleic acid sequences according to the invention can also be used to create antisense oligonucleotides which can be used for regulating the expression of the different JNK3 isoforms. In this respect, the invention also relates to the antisense sequences whose expression in a target cell enables translation of cell mRNAs encoding JNK3xcex94Nxcex11 or JNK3xcex94Nxcex12 or hJNK3Nxcex1139 or else JNK3xcex11 or JNK3xcex12 to be regulated specifically, by hybridizing antisense oligonucleotides with the sequences which are specific to the corresponding mRNAs. Such sequences can, for example, be transcribed, in the target cell, into RNAs which are complementary to the cell mRNAs of the different isoforms of JNK3 and thereby block their translation into protein, in accordance with the technique described in patent EP 140 308. Such sequences can consist of all or part of the nucleotide sequences SEQ ID No. 22, 24 or 26 as transcribed in the reverse orientation.
The invention also makes it possible to produce synthetic or non-synthetic nucleotide probes which are able to hybridize with the above-defined nucleotide sequences. Such probes can be used in vitro as a diagnostic tool for detecting the expression or overexpression of the different JNK3 isoforms or else for detecting genetic anomalies (poor splicing, polymorphism, point mutations, etc.). These probes can also be used for detecting and isolating homologous nucleic acid sequences, which encode peptides as previously defined, from other cell sources, preferably cells of human origin. While the probes of the invention generally comprise at least 17 bases and advantageously at least 300 bases, they can also comprise up to the entirety of one of the abovementioned sequences or their complementary strand. Preferably, these probes are labelled prior to being used. Various techniques known to the skilled person can be employed for this purpose (radioactive, enzymic, etc. labelling).
The nucleotide probes can be employed, in particular by means of PCR, as a diagnostic tool for identifying:
either the isoforms which encode JNK3xcex94Nxcex11 or xcex12, using an oligonucleotide which corresponds to the insertion sequence of 27 nucleotides which is specific to SEQ ID No. 22 and 24 and which is located 5xe2x80x2 of the initiation codon, and an oligonucleotide which is common to all the JNK3 isoforms;
or the isoforms which encode the JNK3s which have been previously described in the literature, using an oligonucleotide which corresponds to the sequence which is located on either side of the insertion region which is present in the sequence of JNK3xcex94Nxcex11 or xcex12, and an oligonucleotide which is common to all the JNK3 isoforms.
The diagnosis can also be effected by means of Northern blotting (Maniatis, T. et al. 1989) in order to identify:
either the isoforms which encode JNK3xcex94Nxcex11 or xcex12, using an oligonucleotide probe which corresponds to the insertion sequence of 27 nucleotides which is specific for SEQ ID No. 22 and 24 and which is located 5xe2x80x2 of the initiation codon;
or the isoforms which encode the JNK3s which have been previously described in the literature, using an oligonucleotide probe which corresponds to the sequence of JNK3 which is located on either side of the insertion region which is present in the sequence of JNK3xcex94Nxcex11 or xcex12.
The present invention also relates to any expression cassette which comprises a nucleic acid as defined above, a promoter which enables it to be expressed and a transcription termination signal.
The invention furthermore relates to any vector which comprises a nucleotide sequence according to the invention or an expression cassette such as defined above. The vector of the invention can, for example, be a plasmid, a cosmid or any DNA which is not encapsidated by a virus, a phage, an artificial chromosome, a recombinant virus, etc. The vector is preferably a plasmid or a recombinant virus.
Those viral vectors in accordance with the invention which may, in particular, be mentioned are the vectors of the adenovirus, retrovirus, adeno-associated virus (AAV), herpes virus or vaccinia virus type. The present application also relates to defective recombinant viruses which comprise a heterologous nucleic acid sequence which encodes a polypeptide according to the invention. Such vectors can be used in gene therapy for treating peripheral traumas such as, in particular, traumas of the spinal cord or retinal degeneration.
The present invention also relates to host cells which are transformed with a nucleic acid which includes a nucleotide sequence or a vector according to the invention. The cell hosts which can be used for producing the polypeptides according to the invention can equally well be eukaryotic hosts or prokaryotic hosts. Suitable eukaryotic hosts which may be mentioned are animal cells, yeasts or fungi. Yeasts which may in particular be mentioned are yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula. Animal cells which may be mentioned are Sf9 insect cells, COS, CHO or C127 cells, human neuroblastoma cells, etc. Fungi which may more particularly be mentioned are Aspergillus ssp. or Trichoderma ssp. The prokaryotic hosts which are preferably employed are the following bacteria E. coli, Bacillus or Streptomyces.
According to a preferred embodiment, the host cells are advantageously represented by strains of recombinant yeasts. Preferably, the host cells comprise at least one sequence or one sequence fragment selected from the nucleotide sequences SEQ ID No. 22 or SEQ ID No. 24 or SEQ ID No. 26 for producing the polypeptides according to the invention.
The present invention also relates to a process for preparing the polypeptides according to the invention, according to which a cell containing a nucleotide sequence according to the invention is cultured under conditions for expressing said sequence and the polypeptide produced is recovered. In this case, the part encoding said polypeptide is generally placed under the control of signals which enable it to be expressed in a cell host. The choice of these signals (promoters, terminators, secretory leader sequence, etc.) can vary depending on the cell host employed. Furthermore, the nucleotide sequences of the invention can be part of a vector, which may be an autonomously replicating vector or an integrating vector. More specifically, autonomously replicating vectors can be prepared by using sequences for autonomous replication in the selected host. Integrating vectors can be prepared, for example, by using sequences which are homologous to particular regions of the host genome and which enable the vector to be integrated by homologous recombination.
The invention also relates to polyclonal or monoclonal antibodies or antibody fragments which are directed against a polypeptide as defined above. Such antibodies can be generated by methods which are known to the skilled person. In particular, these antibodies can be prepared by immunizing an animal against a polypeptide whose sequence is selected from the sequences SEQ ID No. 23 or SEQ ID No. 25 or SEQ ID No. 27 or any fragment or derivative of these sequences and then withdrawing blood and isolating the antibodies. These antibodies can also be generated by preparing hybridomas using the techniques known to the skilled person. The antibodies or antibody fragments according to the invention can, in particular, be used diagnostically for preparing Western blots (Maniatis, T. et al. 1989) which make it possible to identify the different JNK3 protein isoforms in accordance with their molecular weight by using a JNK3-specific antibody to visualise these proteins. These antibodies can also be the starting material for constructing ScFv, which, with the aid of suitable vectors (adenovirus, AAV, retrovirus, etc.), can be used in gene therapy for specifically inhibiting particular JNK3 isoforms in accordance with the technique described in WO94/29446 or in accordance with the technique described by Marasco (Marasco et al., 1997).
According to a preferred embodiment, the invention relates to antibodies which are specific for the JNK3xcex11 and JNK3xcex12 isoforms. These antibodies can, in particular, be monoclonal antibodies which are able to recognize an epitope which is located in the N-terminal part of the JNK3xcex11 and JNK3xcex12 forms. Advantageously, the epitope is an epitope which is contained in the fragment which is delimited by the amino acids which are located in positions 1 and 38 of the JNK3xcex11 and JNK3xcex12 forms; this fragment is depicted in the sequence SEQ ID No. 29. Such antibodies are able to induce a neuroprotective effect by specifically neutralizing the JNK3xcex11 and JNK3xcex12 forms. These antibodies are also able to impart specific properties to the protein which can modify its level of expression, its stability, its catalytic activity, its substrate specificity and its affinity for proteins which are involved in modulating these properties.
The invention also encompasses the antibodies which are derived from the above-defined monoclonal antibodies. Within the meaning of the present invention, derived antibodies are understood as being any molecule which comprises the idiotype of the monoclonal antibodies according to the invention, in particular chimeric antibodies, single-chain antibodies and Fab fragments. Such chimeric antibodies can be obtained using the techniques described by Morrison et al., J. Bacteriol. 159: 870 (1984); Neberger et al., Nature 312: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985), which publications are hereby incorporated into the present application by reference. The Fab fragments which contain the idiotype of the antibodies according to the invention can be generated by any technique known to the skilled person. The invention also relates to single-chain ScFv antibodies which are derived from the above-defined monoclonal antibodies. Such single-chain antibodies can be obtained using the techniques described in patents U.S. Pat. Nos. 4,946,778, 5,132,405 and 5,476,786.
The present invention also relates to a process for identifying compounds which are able to bind to the polypeptides according to the invention. The detection and/or isolation of these compounds can be effected in accordance with the following steps:
a molecule or a mixture containing various molecules, which may possibly not have been identified, is brought into contact with a polypeptide of the invention under conditions which would permit interaction between said polypeptide and said molecule if the latter were to possess an affinity for said polypeptide, and
the molecules which are bound to said polypeptide of the invention are detected and/or isolated.
According to a particular embodiment, such a process makes it possible to identify molecules which are able specifically to block the JNK3xcex94Nxcex11 and JNK3xcex94Nxcex12 isoforms and thereby modulate the processes of neuronal degeneration. These molecules are capable of possessing a neuroprotective activity as a result of specifically inhibiting these isoforms. According to another embodiment, such a process makes it possible to identify specific inhibitors of the JNK3xcex11 and JNK3xcex12 isoforms.
The present invention also relates to processes for identifying compounds which are able to inhibit the activity of the derivatives of JNK3 according to the invention, by measuring the growth of microorganisms transformed with a nucleic acid encoding a polypeptide according to the invention. The detection and/or isolation of these compounds perhaps carried out using a negative screen for drug selection (the drug inhibits the growth of the yeast) or using a positive screen (the drug restores the growth of the yeast).
The latter avoids the selection of antifungal agents which distort the results.
These two screens use a Hog1 mutant expressing an isoform of JNK3. The first is used under hyperosmotic conditions. The second in addition contains the PBS2dd form which hyperstimulates the Hog1 pathway, inducing the death of the yeast under normal conditions. PBS2dd is only toxic if Hog1 is functional or replaced with JNK3.
The present invention therefore relates to a process for identifying compounds which inhibit the growth of microorganisms transformed with a nucleic acid encoding a polypeptide according to the invention, comprising the following steps:
a molecule or a mixture containing various molecules, which may possibly not have been identified, is brought into contact with at least one microorganism transformed with a nucleic acid encoding a polypeptide according to the invention, under conditions which permit the growth of said microorganism, and
the molecules which inhibit the growth of said microorganism are detected and/or isolated.
It also relates to a a process for identifying compounds which permit the growth of microorganisms transformed with a nucleic acid encoding a polypeptide according to the invention, comprising the following steps:
a molecule or a mixture containing various molecules, which may possibly not have been identified, is brought into contact with at least one microorganism transformed with a nucleic acid encoding a polypeptide according to the invention, under conditions which inhibit the growth of said microorganism, and
the molecules which permit the growth of said microorganism are detected and/or isolated.
This microorganism may be a yeast containing an inactive Hog1 gene and expressing one of the novel isoforms of JNK3 according to the invention, such as that described in the examples which follow, or any other microorganism having equivalent properties. This yeast grows under hyperosmotic conditions. Products which inhibit these novel isoforms of JNK3 are able to inhibit or slow down the growth of this strain under hyperosmotic conditions.
According to another embodiment, a mutated form of PBS2: PBS2DD (Wurgler-Murphy et al. 1997) which has a toxic effect on the growth of the yeast is expressed in the strain indicated above. This toxicity depends on the activity of the protein kinase located downstream which, according to the present invention, is a novel isoform of JNK3. Products which inhibit these novel isoforms of JNK3 are, in this case, able to inhibit the toxic effect of mutated PBS2 and thus to permit restoration of the growth of this strain.
The invention also relates to compounds or ligands which are able to inhibit the activity of the polypeptides according to the invention and which can be obtained using one of these microorganisms as a screening tool.
The invention also relates to compounds or ligands which are able to bind to the polypeptides according to the invention and which can be obtained using one of the above-defined processes.
The invention also relates to the use of a compound or ligand which has been identified and/or obtained using one of the above-described processes as a medicament. This is because such compounds can be used for preventing, ameliorating or treating various pathologies brought about by neuronal degeneration, among which may be mentioned Alzheimer""s diseases, Huntington""s disease and Parkinson""s disease, senile dementias and dementias due to AIDS, cranial traumas, anoxias, hypoxias and cerebral oedemas.
The invention also relates to any pharmaceutical composition which comprises, as the active principle, at least one compound or ligand as defined above.
The polypeptides of the invention are also useful for identifying other partners involved in the mechanisms of neuronal degeneration or in the field of cardiac ischaemia by searching for and identifying molecules which interact in vivo with these polypeptides. In this respect, the present invention also relates to a process for identifying partner polypeptides which are specific for the different natural isoforms of JNK3. The specific partner polypeptides can be partner polypeptides which are specific for the JNK3xcex11xcex94N or JNK3xcex12xcex94N or hJNK3xcex1139 forms or partner polypeptides which are specific for the JNK3xcex11 and JNK3xcex12 forms. The partner polypeptides which are specific for the different isoforms of JNK3 can be identified by the double hybrid cloning technique using the techniques described by Fields et al. 1994; the bait protein can, in particular, be the entire JNK3xcex94Nxcex11 or JNK3xcex94Nxcex12 protein or the N-terminal 38 amino acid extension of JNK3 which is lacking in the JNK3xcex94N isoforms.
Other advantages of the present invention will be apparent from reading the examples which follow and which should be regarded as being illustrative and not limiting.